The long term goal of this proposal is to develop a molecular understanding of the mechanisms of sweet receptor activation by combining experimental and computational approaches. The taste preference for sweet compounds allows animals to seek out high carbohydrate energy sources to exploit for food. The sweet receptor is composed of two type 1 taste receptor monomers (T1R2 plus T1R3), apparently as a heterodimer. This proposal uses mutagenesis of the sweet receptor, expression in HEK 293 cells, calcium imaging, bioluminescent-resonance-energy-transfer and surface expression of receptors, to probe the sweet receptor's interaction with ligands. Aim 1 uses computational approaches to homology model the large extracellular domain of the heterodimer, using as template the crystal structure of the extracellular domain of mGluRl , another member of this family of receptors. The resulting homology models are tested and refined by mutagenesis of residues in T1R2+T1R3 predicted to form the dimerization interface, and then the expressed receptors are assayed for responses to sweet ligands and formation of heterodimers. The resulting optimized models will be useful to explain effects of mutations on ligand-induced activity in subsequent Aims. Aim 2 seeks to discover T1R2 residues that influence ligand-receptor interaction and receptor activation. Aim 2a uses the alignment of the TIR s with mGluRl to choose potential ligand-interacting residues in T1R2, then mutate them to discover their effects on receptor responses to sweet ligands. Aim 2b employs differences in species-specific taste perception, and chimeric human/mouse T1R2 receptors to track portions of the receptor responsible for human-like responses to sweeteners. Aim 2c uses mutagenesis to scan the surface-accessible arginines and lysines that might interact with the brazzein dipole. Mutated receptors are expressed, assayed for loss of responsiveness toward brazzein, then brazzein mutants are tested for the ability to compensate for receptor mutations. Aim 3 follows up on our recent observation that two residues in the cysteine-rich linker region of human T1R3 are essential for receptor responses to brazzein. We have proposed makin g additional mutations in this region to identify and characterize those residues that enable the human receptor to respond to brazzein. The knowledge gained from these studies will provide a working model for sweet receptor activity that may lead to the design of superior artificial sweeteners. Our molecular studies of the sweet receptor may shed light on transduction mechanisms common to other members of this family of receptors, such as the calcium-sensing receptor, which regulates calcium metabolism, and the metabotropic glutamate receptors, which are involved in multiple neurological responses.